mTOR contributes to endothelium-dependent vasorelaxation by promoting eNOS expression and preventing eNOS uncoupling

Clinically used inhibitors of mammalian target of rapamycin (mTOR) negatively impacts endothelial-dependent vasodilatation (EDD) through unidentified mechanisms. Here we show that either the endothelium-specific deletion of Mtor to inhibit both mTOR complexes, or depletion of Raptor or Rictor to disrupt mTORC1 or mTORC2, causes impaired EDD, accompanied by reduced NO in the serum of mice. Consistently, inhibition of mTOR decreases NO production by human and mouse EC. Specifically, inhibition of mTORC1 suppresses eNOS gene expression, due to impairment in p70S6K-mediated posttranscriptional regulation of the transcription factor KLF2 expression. In contrast to mTORC1 inhibition, a positive-feedback between MAPK (p38 and JNK) activation and Nox2 upregulation contributes to the excessive generation of reactive oxygen species (ROS), which causes eNOS uncoupling and decreased NO bioavailability in mTORC2-inhibited EC. Adeno-associated virus-mediated EC-specific overexpression of KLF2 or suppression of Nox2 restores EDD function in endothelial mTORC1- or mTORC2-inhibited mice.


Supplementary Figure 2.
Inhibition of mTORC1 decreased KLF2 expression. a-b, HAEC were exposed to 1nM rapamycin (rapa, b) or 10nM torin 1 (torin, c) for 1h prior to NO measurement by EPR technique. Shown to the right are presentative EPR spectra images. c, HAEC were treated with 1nM rapa prior to Western blot analysis. d, HAEC were transfected with KLF2-targeting siRNA prior to quantitative PCR (n=4). ef, quantitative PCR (n=6) and Western blot analysis were performed after transfection of pcDNA3.1 EIF4EBP-1 into HAEC. Error bars correspond to standard error of the mean (SEM). *p<0.05; **p<0.01 vs. ctrl or sictrl or sham; one sample t test.

Supplementary Figure 3
Supplementary Figure 3. Prolonged treatment with rapamycin caused ROS accumulation. a, HAEC were treated with 1nM rapa for 48h prior to DHE staining and measurement of ROS with flow cytometry (n=5). b, Serum ROS levels were measured in mice administered with vehicle (ctrl) or rapamycin (rapa) with chemiluminescent assay (n=5). c, After treated with 500nM L-NAME for 0.5h (n=4), HAEC were submitted to NO assay to confirm the inhibitory effect of L-NAME on NOS. d, HAEC were treated with 1nM rapa or 10nM torin for 1h prior to Western blot analysis and quantification of iNOS protein (n=5). e, After 1h pretreatment with 20µM Nox inhibitor Apocynin (Apo) (n=3), HAEC were incubated with 10nM torin for 1h prior to ERP measurement of the culture medium. The results confirmed that with chromogenic reaction assay. f, HAEC were treated with 5µM Gö-6976 (Gö) or 5µM Rottlerin (Rott) for 1h before incubation with or without 10nM torin for 1h. ROS production was then evaluated with flow cytometry (n=3-4). g, The phosphorylation of p38 and JNK1/2 was examined by Western blotting after transfection of siRNA into HAEC. Shown are representative images of four experiments (n=4). Error bars correspond to standard error of the mean (SEM). *p<0.05 vs. ctrl; one-sample t-test, a-b, e; unpaired two-tailed t test, c; RM ANOVA followed by Dunnett's test, d, f.

Supplementary Figure 4
Supplementary Figure 4. Rptor EC-/mice were infected with empty AAV (AAV-EGFP) or AAV-Klf2 at a dose of 3×10 11 vg/mouse while Rictor EC-/mice infected with empty AAV (AAV-EGFP) or AAV-shNox2. a, Fluorescence microscopy analysis of the aorta sections confirmed ICAM2 promoter-driven EC-specific expression of EGFP as evidenced by its co-localization with CD31. b, Infection of Rptor EC-/mice with AAV-Klf2 successfully increased the expression of eNOS specifically in the EC. c, Nox2 expression in EC was inhibited by infection of AAV-shNox2. Scale bar, 50 μm. d, Representative images of transcutaneous ultrasound assay demonstrated the improved vascular vasodilatation function of Rptor EC-/mice infected with AAV-Klf2. Scale=0.5 mm. e, Rptor EC-/mice were i.v. injected with AAV-GFP or AAV-Klf2 at 3×10 11 vg. Serum NO was evaluated by EPR assay (n=4). Shown to the right are representative EPR spectra. f, Representative images of transcutaneous ultrasound assay demonstrated the improved vascular vasodilatation function of Rictor EC-/mice infected with AAV-shNox2. Scale=0.5 mm. g, Rictor EC-/mice were injected with 3×10 11 vg AAV-GFP or AAV-shNox2 followed by evaluation of NO by EPR assay (n=4). *p<0.05; one-sample t-test, e, g.
Original Western Blot images for Ⅰ) Flag-KLF2, GAPDH, Ⅱ)eNOS in EC isolated from wild type mice were infected with empty AAV (AAV-GFP) or Klf2-expressing adenoassociated virus (AAV-Klf2) at a multiplicity of infection (MOI) of 10 5 vg/cell for 72h, then treated with or without 1nM rapa for 1h. Bands showing in Figure 7c are indicated in red rectangles.
Original Western Blot images for Nox2, β -actin in murine EC infected with AAV containing two tandem mouse Nox2-targeting sequences (AAV-shNox2). Bands showing in Figure 7d are indicated in red rectangles.